Knockdown of sulfotransferase 2B1 suppresses cell migration, invasion and promotes apoptosis in ovarian carcinoma cells via targeting annexin A9.

BACKGROUND: Sulfotransferase family 2B member 1 (SULT2B1) has been reported to play oncogenic role in many types of cancers. Nevertheless, the role that SULT2B1 played in ovarian cancer (OC) and the hidden molecular mechanism is obscure. METHODS: Expression of SULT2B1 in OC was analyzed by GEPIA database. qRT-PCR and western blot (WB) was applied for the appraisement of SULT2B1 and Annexin A9 (ANXA9) in OC cell lines. The capabilities of cells to proliferate, migrate and invade were assessed with CCK-8 assay, wound healing assay, along with transwell assay. Cell apoptotic level was estimated utilizing flow cytometry. WB was employed for the evaluation of migration- and apoptosis-related proteins. Bioinformatic analysis and co-immunoprecipitation were used to predict and verify the combination of SULT2B1 and ANXA9. RESULTS: The data showed that SULT2B1 and ANXA9 were upregulated in OC cells. SULT2B1 depletion suppressed the proliferative, migrative, and invasive capabilities of SKOV3 cells but facilitated the cell apoptosis. SULT2B1-regulated ANXA9 expression and were proved to bind to ANXA9. Additionally, ANXA9 deficiency exhibited the same impacts on cell migrative, invasive capability and apoptotic level as SULT2B1 silencing. Moreover, ANXA9 overexpression reversed the inhibitory impacts of SULT2B1 silencing on the proliferative, migrative, invasive, and apoptotic capabilities of SKOV3 cells. CONCLUSION: In summary, SULT2B1 silencing repressed OC progression by targeting ANXA9.

[Expression of CD44, CD87 and CD123 in Acute Leukemia and Its Correlation with Cellular Immunity].

OBJECTIVE: To investigate the expression of CD44, CD87 and CD123 in acute leukemia and its correlation with cellular immune markers. METHODS: A total of 166 patients with acute leukemia (AL) admitted from May 2014 to February 2017 were enrolled in AL groups. Among these patients, 100 patients suffered from acute myeloid leukemia, 50 patients suffered from acute lymphoid leukemia, and 16 patients showed B/medullary phenotype. At the same time 50 patients with non-acute leukemia were enrolled in the control group. 5 ml of fasting venous blood collected from the patients in each group, and the percentage of CD44, CD87 and CD123 cells was determined by three-color flow cytometry. Symptomatic chemotherapy was given to the patients with confirmed acute leukemia, and the remission was evaluated after 2 treatmen courses. The Complete remission (CR) was recorded and the percentage of CD44, CD87 and CD123 cells under different curative efficacy were recorded. The correlation of the prognosis patients with CD44, CD87 and CD123 was analyzed by SPSS Pearson correlation analysis software. RESULTS: The positive rates of CD44, CD87 and CD123 in AL group were all higher than those in the control group (P＜0. 05). The positive rates of CD44 and CD123 in acute myeloid leukemia group were higher than those in acute lymphoblastic leukemia group and B/myeloid phenotype group (P＜0. 05). The positive rate of CD44 in acute lymphoid leukemia group was higher than that in B/medullary double phenotype group (P＜0.05). The treatment in the patients of AL group was successfully completed. 132 patients reachel to CR and 34 patients to PR+NR after 2 courses. The positive rates of CD44, CD87 and CD123 in CR patients were lower than those in PR+NR patients (P＜0.05). The results of SPSS Pearson correlation analysis showed that the prognosis of patients with acute leukemia negatively correlated with CD44 and CD87 (P＜0.05). CONCLUSION: The expression of CD44, CD87 and CD123 in different phenotype of acute leukemia are different, which correlateds with prognosis. The determination of CD44, CD87 and CD123 can be used to evaluate the prognosis of patients for the reference of clinical treatment.

A Novel Glycoside Hydrolase Family 113 Endo-ß-1,4-Mannanase from Alicyclobacillus sp. Strain A4 and Insight into the Substrate Recognition and Catalytic Mechanism of This Family.

Few members of glycoside hydrolase (GH) family 113 have been characterized, and information on substrate recognition by and the catalytic mechanism of this family is extremely limited. In the present study, a novel endo-ß-1,4-mannanase of GH 113, Man113A, was identified in thermoacidophilic Alicyclobacillus sp. strain A4 and found to exhibit both hydrolytic and transglycosylation activities. The enzyme had a broad substrate spectrum, showed higher activities on glucomannan than on galactomannan, and released mannobiose and mannotriose as the main hydrolysis products after an extended incubation. Compared to the only functionally characterized and structure-resolved counter part Alicyclobacillus acidocaldarius ManA (AaManA) of GH 113, Man113A showed much higher catalytic efficiency on mannooligosaccharides, in the order mannohexaose ≈ mannopentaose > mannotetraose > mannotriose, and required at least four sugar units for efficient catalysis. Homology modeling, molecular docking analysis, and site-directed mutagenesis revealed the vital roles of eight residues (Trp13, Asn90, Trp96, Arg97, Tyr196, Trp274, Tyr292, and Cys143) related to substrate recognition by and catalytic mechanism of GH 113. Comparison of the binding pockets and key residues of ß-mannanases of different families indicated that members of GH 113 and GH 5 have more residues serving as stacking platforms to support -4 to -1 subsites than those of GH 26 and that the residues preceding the acid/base catalyst are quite different. Taken as a whole, this study elucidates substrate recognition by and the catalytic mechanism of GH 113 ß-mannanases and distinguishes them from counterparts of other families.

High level expression of a novel family 3 neutral ß-xylosidase from Humicola insolens Y1 with high tolerance to D-xylose.

A novel ß-xylosidase gene of glycosyl hydrolase (GH) family 3, xyl3A, was identified from the thermophilic fungus Humicola insolens Y1, which is an innocuous and non-toxic fungus that produces a wide variety of GHs. The cDNA of xyl3A, 2334 bp in length, encodes a 777-residue polypeptide containing a putative signal peptide of 19 residues. The gene fragment without the signal peptide-coding sequence was cloned and overexpressed in Pichia pastoris GS115 at a high level of 100 mg/L in 1-L Erlenmeyer flasks without fermentation optimization. Recombinant Xyl3A showed both ß-xylosidase and α-arabinfuranosidase activities, but had no hydrolysis capacity towards polysaccharides. It was optimally active at pH 6.0 and 60°C with a specific activity of 11.6 U/mg. It exhibited good stability over pH 4.0-9.0 (incubated at 37°C for 1 h) and at temperatures of 60°C and below, retaining over 80% maximum activity. The enzyme had stronger tolerance to xylose than most fungal GH3 ß-xylosidases with a high Ki value of 29 mM, which makes Xyl3A more efficient to produce xylose in fermentation process. Sequential combination of Xyl3A following endoxylanase Xyn11A of the same microbial source showed significant synergistic effects on the degradation of various xylans and deconstructed xylo-oligosaccharides to xylose with high efficiency. Moreover, using pNPX as both the donor and acceptor, Xyl3A exhibited a transxylosylation activity to synthesize pNPX2. All these favorable properties suggest that Xyl3A has good potential applications in the bioconversion of hemicelluloses to biofuels.

Interplay between mTOR and STAT5 signaling modulates the balance between regulatory and effective T cells.

BACKGROUND: Immune response outcome, inflammation or tolerance, often depends on the balance between regulatory T cells (Tregs) and effective T cells (Teffs). Rapamycin (Rapa) has been reported to selectively expand Tregs and promote de novo generation of foxp3(+) Tregs, suggesting its potential role in inducing tolerance. But the mechanism by which Rapa regulating the Treg-Teff balance is yet to be understood. METHODS: Mouse CD4(+)CD25(-) Teffs and CD4(+)CD25(+) Tregs are sorted by MACS. These T cell subsets were labeled with CFSE and cultured with anti-CD3/CD28 Ab±IL-2 for 6 days. Two rounds of stimulation of 3 days each were performed. Rapa or Jak Inhibitor I was added to the culture when indicated. Cells were harvested after each round of stimulation. CFSE dilution, FOXP3, miR-155 expression and the signaling via the mTOR and STAT5 pathways were determined. And miR-155-mimic and miR-155-antagomir were transfected into purified CD4(+) T cells respectively to detect miR-155 role in regulating STAT5 signaling. RESULTS: Firstly, we confirmed that the effect of Rapa on proliferating T cells is time-dependent: it reduces both Teffs and Tregs proliferation at early stage, but selectively promotes Tregs proliferation after second round of stimulation. Then we found there is direct interaction between mTOR and STAT5 signaling, and this interaction explained the time-dependent effect of Rapa and may participate in deciding Teff-Treg balance: mTOR inhibition up-regulated the expression of phos-STAT5 in both proliferating Tregs and Teffs via miR-155. And foxp3 is the down streaming target of phos-STAT5, thus Rapa could maintain expanded Tregs function and promote de novo generation of foxp3(+) Tregs. However, the phos-4E-BP1 expression pattern is different in proliferating Tregs and Teffs. 4E-BP1 is the common target of mTOR and STAT5 signaling, and plays a key role in cell proliferation. Rapa inhibits phos-4E-BP1 expression in both Tregs and Teffs at early stage of proliferation, but selectively raises its expression in Tregs after second round of stimulation. This may explains why Rapa inhibits Teffs growth, but delays Tregs proliferation. CONCLUSION: Together, these findings indicate that the dynamic interaction between mTOR and STAT5 signaling modulates the reciprocal differentiation of the effective and regulatory T cells, and differently affect their proliferation activity. This provides a new insight of how Treg-Teff balance is regulated.

Over-expression of microRNA-494 up-regulates hypoxia-inducible factor-1 alpha expression via PI3K/Akt pathway and protects against hypoxia-induced apoptosis.

BACKGROUND: Hypoxia-inducible factor-1 alpha (HIF-1α) is one of the key regulators of hypoxia/ischemia. MicroRNA-494 (miR-494) had cardioprotective effects against ischemia/reperfusion (I/R)-induced injury, but its functional relationship with HIF-1α was unknown. This study was undertaken to determine if miR-494 was involved in the induction of HIF-1α. RESULTS: Quantitative RT-PCR showed that miR-494 was up-regulated to peak after 4 hours of hypoxia in human liver cell line L02. To investigate the role of miR-494, cells were transfected with miR-494 mimic or miR-negative control, followed by incubation under normoxia or hypoxia. Our results indicated that overexpression of miR-494 significantly induced the expression of p-Akt, HIF-1α and HO-1 determined by qRT-PCR and western blot under normoxia and hypoxia, compared to negative control (p < 0.05). While LY294002 treatment markedly abolished miR-494-inducing Akt activation, HIF-1α and HO-1 increase under both normoxic and hypoxic conditions (p < 0.05). Moreover, apoptosis detection using Annexin V indicated that overexpression of miR-494 significantly decreased hypoxia-induced apoptosis in L02 cells, compared to control (p < 0.05). MiR-494 overexpression also decreased caspase-3/7 activity by 1.27-fold under hypoxia in L02 cells. CONCLUSIONS: Overexpression of miR-494 upregulated HIF-1α expression through activating PI3K/Akt pathway under both normoxia and hypoxia, and had protective effects against hypoxia-induced apoptosis in L02 cells. Thus, these findings suggested that miR-494 might be a target of therapy for hepatic hypoxia/ischemia injury.

Adoptive infusion of tolerance dendritic cells prolongs survival of small intestine allografts in rats: systematic review and meta-analysis.

BACKGROUND: Postoperative infections and rejection are the main limiting factors of small intestine allograft survival. In this study, we performed a systematic review and meta-analysis to review rat small intestine allograft survival following infusion of tolerance dendritic cells (Tol-DCs) induced by different methods. METHODS: Relevant publications were searched from PubMed database and EMbase database. Meta-analysis was performed using RevMan 5.0 software. We chose allograft survival, mixed leukocyte reaction, Th1/Th2 differentiation, Treg induction, and cytotoxic T lymphocyte activity as the outcomes by which to examine possible mechanisms that promote survival. RESULTS: Eleven suitable articles were identified and assessed. Tol-DCs induced by four methods all prolonged allograft survival. The difference in survival time between the Tol-DC group and the control group was indicated by SMD as follows: drug intervention (SMD = 3.02, 95% CI 1.16 to 4.88, P = 0.001), gene modification (SMD = 2.43, 95% CI 1.77 to 3.10, P < 0.00001), imDC (SMD = 1.76, 95% CI 0.90 to 2.62, P < 0.0001), cytokine induction (SMD = 1.68, 95% CI 0.40 to 2.96, P = 0.01). Tol-DCs were also synergistic with immunosuppressive drugs or costimulation inhibitors, but no immune tolerance was observed. A single-dose intravenous injection of 5×10(6) to 6×10(6) Tol-DCs showed the highest allograft survival. Possible mechanisms included donor-specific T-cell hyporesponsiveness and Th2 differentiation. CONCLUSIONS: Our results demonstrated that Tol-DCs induced by four methods prolong rat small intestine allograft survival. Intravenous infusion of 5×10(6) to 6×10(6) Tol-DCs was the optimum dose in rat small intestine transplantation. Immunosuppressive or costimulatory blockade was synergistic with Tol-DC on graft survival. Additional high-quality studies with larger sample sizes are needed to better investigate small intestinal graft longer term survival.

Adoptive transfusion of tolerance dendritic cells prolongs the survival of skin allografts in mice: a systematic review.

OBJECTIVE: We aim to systematically review adoptive transfusion of tolerogenic dendritic cells (Tol-DCs) induced by different ways to affect skin allograft survival in mice. METHODS: We searched PubMed and EMbase for relevant studies and evaluated the quality of included ones. Taking skin allograft survival time as endpoint outcome, we displayed outcomes of each group using one forest map and dissected possible mechanisms underlying survival prolongation. RESULTS: We included 21 studies, which reported four methods of inducing Tol-DCs with different extents of average allograft survival prolongation: skin allograft survival time was prolonged (the drug intervention group, 63.08 ± 42.92 days, 4.6 folds to control; the cytokine induction group: 26.17 ± 16.20 days, 1.8 folds; the gene modification group: 14.65 ± 17.89 days, 1.5 folds; other derivation group 9.63 ± 24.38 days, 0.5 fold). Possible mechanisms underlying survival prolongation included induction of donor-specific T cell hyporesponsiveness, reduction of cytotoxicity against allografts, Th0 skewing to Th2, and generation or expansion of Treg. Infusion of Tol-DCs in combination with immunosuppressive agents or costimulatory blockade contributed to longer prolongation. Compared to MiHA mismatch, MHCI/II mismatch was a much more important factor to cause skin allograft rejection. CONCLUSION: For MHC or MiHA mismatched, allogeneic skin transplants inbred recipients, adoptive transfusion of Tol-DCs induced by 4 methods prolong skin allograft survival to different extents. Drug intervened Tol-DCs works best. Immunosuppressive agents and/or co-stimulatory blockade contribute to better outcomes. Yet more rigorous studies with larger sample size are needed and more attention to mechanisms should be paid.

Adoptive infusion of tolerogenic dendritic cells prolongs the survival of pancreatic islet allografts: a systematic review of 13 mouse and rat studies.

OBJECTIVE: The first Phase I study of autologous tolerogenic dendritic cells (Tol-DCs) in Type 1 diabetes (T1D) patients was recently completed. Pancreatic islet transplantation is an effective therapy for T1D, and infusion of Tol-DCs can control diabetes development while promoting graft survival. In this study, we aim to systematically review islet allograft survival following infusion of Tol-DCs induced by different methods, to better understand the mechanisms that mediate this process. METHODS: We searched PubMed and Embase (from inception to February 29(th), 2012) for relevant publications. Data were extracted and quality was assessed by two independent reviewers. We semiquantitatively analyzed the effects of Tol-DCs on islet allograft survival using mixed leukocyte reaction, Th1/Th2 differentiation, Treg induction, and cytotoxic T lymphocyte activity as mechanisms related-outcomes. We discussed the results with respect to possible mechanisms that promote survival. RESULTS: Thirteen articles were included. The effects of Tol-DCs induced by five methods on allograft survival were different. Survival by each method was prolonged as follows: allopeptide-pulsed Tol-DCs (42.14 ± 44 days), drug intervention (39 days), mesenchymal stem cell induction (23 days), genetic modification (8.99 ± 4.75 days), and other derivation (2.61 ± 6.98 days). The results indicate that Tol-DC dose and injection influenced graft survival. Single-dose injections of 10(4) Tol-DCs were the most effective for allograft survival, and multiple injections were not superior. Tol-DCs were also synergistic with immunosuppressive drugs or costimulation inhibitors. Possible mechanisms include donor specific T cell hyporesponsiveness, Th2 differentiation, Treg induction, cytotoxicity against allograft reduction, and chimerism induction. CONCLUSIONS: Tol-DCs induced by five methods prolong MHC mismatched islet allograft survival to different degrees, but allopeptide-pulsed host DCs perform the best. Immunosuppressive or costimulatory blockade are synergistic with Tol-DC on graft survival. Multiple injections are not superior to single injection. Yet more rigorously designed studies with larger sample sizes are still needed in future.

Adoptive transfusion of tolerance dendritic cells prolongs the survival of cardiac allograft: a systematic review of 44 basic studies in mice.

BACKGROUND AND OBJECTIVE: Tolerogenic DCs (Tol-DCs), a group of cells with imDC phenotype, can stably induce T cells low-reactivity and immune tolerance. We systematically reviewed the adoptive transfusion of Tol-DCs induced by different ways to prolong cardiac allograft survival and its possible mechanism. METHOD: MEDLINE (1966 to March 2011), EMbase (1980 to March 2011), and ISI (inception to March 2011) were searched for identification of relevant studies. We used allogeneic heart graft survival time as endpoint outcome to analyze the effect of adoptive transfusion of Tol-DC on cardiac allograft. By integrating studies' information, we summarized the mechanisms of Tol-DC in prolonging cardiac grafts. RESULTS: Four methods were used to induce Tol-DC in all of the 44 included studies including gene-modified, drug-intervened, cytokine-induced, and other-derived (liver-derived & spleen-derived) DCs. The results showed that all types of Tol-DC can effectively prolong graft survival, and the average extension of graft survival time for each group was as follows: 22.02 ± 21.9 days (3.2 folds to control group) in the gene modified group, 25.94 ± 16.9 days (4.3 folds) in the drug-intervened groups, 9.00 ± 8.13 days (1.9 folds) in the cytokine-induced group, and 10.69 ± 9.94 days (2.1 folds) in the other-derived group. The main mechanisms of Tol-DCs to prolong graft survival were as follows: (1) induceT-cell hyporeactivity (detected by MLR); (2) reduce the effect of cytotoxic lymphocyte (CTL); (3) promote Th2 differentiation; (4) induce Treg; (5) induce chimerism. CONCLUSION: For fully MHC mismatched allogeneic heart transplant recipients of inbred mouse, adoptive transfusion of Tol-DC, which can be gene-modified, drug-intervened, cytokine-induced, spleen-derived or liver-derived, can clearly prolong the survival of cardiac allograft or induce immune tolerance. Gene-modified and drug-induced Tol-DC can prolong graft survival most obviously. Having better reliability and stability than drug-induction, gene-modification is the best way to induce Tol-DCs at present. One-time intravenous infusion of 2 × 10(6) Tol-DC is a simple and feasible way to induce long-term graft survival. Multiple infusions will prolong it but increase the risk and cost. Adoptive transfusion of Tol-DC in conjunction with immunosuppressive agents may also prolong the graft survival time.